1. Field of the Invention
The present invention generally relates to nucleic acid molecules of Bacillus anthracis. The nucleic acid molecules may be used in nucleic acid assays.
2. Description of the Related Art
Bacillus anthracis is a spore-forming gram-positive bacterium well known for its recent use as a bioterrorist agent. Identification of B. anthracis can be done clinically utilizing gram stain, colony morphology, and various biochemical tests. See Logan & Turnbull (2003) In Manual of Clinical Microbiology. American Society of Microbiology, Washington D.C. However, these methods are time consuming and more rapid tests, such as polymerase chain reaction (PCR), have been employed to detect B. anthracis in clinical samples. See Oggioni, et al. (2002) J. Clin. Microbiol. 40:3956-3963.
Real-time PCR is preferred over conventional PCR methods for the identification of organisms because it is fast, less labor intensive, and adds the specificity of a probe. Real-time PCR assays have been used to identify anthracis based on virulence genes associated with the toxin-encoding plasmid (pX01) and capsule-encoding plasmid (pX02). See Higgins, et al. (2003) Appl. Environ. Microbiol. 69:593-599; Oggioni, et al. (2002) J. Clin. Microbiol. 40:3956-3963; and Patra, et al. (2002) Ann. N.Y. Acad. Sci. 969:106-111. While the presence of both pX01 and pX02 is needed to give B. anthracis its virulence, it is conceivable that these plasmids could be passed to its genetic neighbors with unknown implications. Thus, a chromosomal marker for use in nucleic acid based assays such as real-time PCR assays is more desirable.
Unfortunately, past attempts at developing a chromosomal real-time PCR assay have failed due to the close genetic relationship of Bacillus species. B. anthracis, Bacillus cereus, and Bacillus thuringiensis have very little variability and are genetically indistinguishable using multilocus enzyme electrophoresis. See Helgason, et al. (2000) Appl. Environ. Microbiol. 66:2627-2630. Recent work using rep-PCR has shown that the previously listed species of Bacillus as well as Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis do have some genetic differences. See Cherif, et al. (2003) J. Appl. Microbiol. 94:1108-1119. A real-time PCR assay based on the chromosomal rpoB gene has been developed and used, however, it targets a region that is variable between Bacillus species, therefore, the specificity of the assay is dependent on PCR conditions and specific primers and probes. See Drago, et al. (2002) J. Clin. Microbiol. 40:4399; and Qi, et al. (2001) Appl. Environ. Microbiol. 67:3720-3727.
Thus, a need still exists for a unique chromosomal nucleotide sequence in B. anthracis for use in nucleic acid based assays such as real-time PCR assays.